Part:BBa_K2923018:Experience

Introduction

To validate the designed constructs we decided to test two different aptazymes : Theophylline switch-off (stop auto-cleavage in presence of ligand) and Guanine switch-on (active auto-cleavage in presence of ligand). For negative control, we choose an inactive form of aptazyme : mutation at ribozyme domain. iGEM Strasbourg designed construct require the use of aptazyme linked to MS2 and PP7 stem-loops. The following results described MS2-MS2-Aptazyme-PP7-PP7 activity in in-vitro and in-vivo assays.

In-vitro assay of BBa_K2923018

In order to check MS2-MS2-Theophylline-PP7-PP7 cleavage activities, in-vitro transcription products were analyzed by 2% agarose gel with urea (Figure 1).

Figure 1: Aptazyme catalytic activity assay. Theophylline aptazyme were transcribed in vitro for 1h30. RNA was analyzed in an 8% polyacrylamide gel with urea. The inactive form of Theophylline aptazyme represent the whole RNA and correspond to band of 94 pb. Clived aptazyme represent the two parts of the aptazyme Theophylline (18 pb + 76 pb).

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